This invention relates to a method of purifying human plasma procoagulant protein Factor VIII:C from Factor VIII concentrates.
For several decades there has been considerable interest in the human plasma Factor VIII procoagulant protein because of its value in the treatment of hemophilia A. Invivo or circulating Factor VIII is known to be a large complex composed of two different proteins, a carrier protein (von Willibrand factor) and the procoagulant protein Factor VIII:C which are linked together by noncoavalent bonds. Von Willibrand factor interacts with platelets in a way that promotes primary hemostasis and is usually designated Factor VIII R (ristocetin cofactor or von Willebrand antigen). Hemophilia A patients, that is those patients suffering from classical hemophilia, lack functional Factor VIII:C and require infusion of Factor VIII:C in order to overcome bleeding episodes.
Factor VIII (also known as the Antihemophilic Factor A or AHF) is commercially available as a lyophilized sterile dry powder which is reconstituted with sterile distilled water or sterile physiological saline for infusion into a hemophilia A patient.
A number of processes exist for the preparation of plasma concentrates for clinical use which contain the Factor VIII complex or parts thereof together with other plasma constituents. The concentrates have been a boon to hemophiliac A patients; unfortunately, the specific activity of the concentrates is low and therefore great volumes of constitute solution must be injected into the patient for treatment of hemophilia A. A number of methods have been developed for preparing concentrates with higher specific activities; unfortunately, most of these methods have very low recovery rates and are costly. Because of the low recovery rates and high cost of the methods, these methods have not been utilized on a commercial scale to produce Factor VIII concentrates having a high activity.
U.S. Pat. No. 3,920,625 to L. O. Andersson et al discloses a 12 step process for isolating Factor VIII (AHF) and the B-Factor from blood plasma. An essential step according to the inventors is the adsorption of at least one of these factors, in a liquid system onto a water-soluble gel matrix thus leaving the other factor in the supernatant liquid.
U.S. Pat. No. 4,069,216 to E. Shanbrom discloses a method of selectively removing the fibrinogen from a solution of blood plasma or plasma by treating the buffered plasma with polyol and maintaining the resulting mixture at a temperature of from about 0.degree. C. to about 5.degree. C. until precipitation of the fibrinogen occurs.
U.S. Pat. No. 4,397,841 to J. H. Johnson is directed to a method of concentrating Factor VIII by fractionating plasma with a sequence of adsorption steps employing two different water-insoluble cross-linked polyelectrolyte copolymers, each in the presence of exogenous heparin. In the first adsorption step, Factor IX complex is removed from the supernatant liquid. In the second adsorption step, Factor VIII is adsorbed and recovered from the adsorbent after separation of the adsorbent from the supernatant liquid. Recoveries of 40-70% of the original Factor VIII are reported.
U.S. Pat. No. 4,348,315 to E. G. Birger Blomback et al discloses a process for the purification and/or concentration of Factor VIII complex by dissolving the Factor VIII complex, including impurities, in a solution containing at least 1.5M glycine, having a pH of about 6.3-7.8 and maintaining the resulting mixture at a temperature of from 15.degree. C. to 40.degree. C. to precipitate out impurities leaving the Factor VIII in the supernatant liquid.
U.S. Pat. No. 3,637,489 to W. Haller is directed to a process for the separation of blood components using steric chromatography. Haller discloses that it is known to separate serum proteins with the aid of gel filtration, also called gel chromatography, steric chromatography, exclusion chromatography or gel permeation chromatography. This method utilizes porous auxilliary substances which have pores of such dimensions that molecules to be separated diffuse into and out of the pores at different velocities or are prohibited from entering the pores. Haller further discloses they can be used to fractionate serum proteins on a laboratory research scale but cannot be used for large-scale fractionation and particularly if the product should qualify for medical applications.
U.S. Pat. No. 3,657,116 to W. Haller discloses a process for the separation of blood components by fractionating the blood plasma or serum in a chromatographic column filled with porous glass particles.
U.S. Pat. No. 4,108,603 to F. E. Regnier et al discloses a method of analysis of hemoglobin variance by high speed liquid chromatography at high pressures employing a bonded phase inorganic support column. The bonded phase support preferably includes ion-exchange groups bonded through a carbohydrate intermediate to controlled porosity glass.
The paper by Phillip J. Fay et al, "Purification and Characterization of a Highly Purified Human Factor VIII Consisting of a single Type of Polypeptide Chain", Proc. Natl. Acad. Sci., Vol. 79, pages 7200-7204, December, 1982 discloses a procedure for purification of human Factor VIII from plasma which utilizes calcium dissociation and differential size and charge chromatography. The human Factor VIII was purified over 350,000 fold (relative to plasma) from a commercial Factor VIII concentrate with a final yield of 13%. The process involves seven steps: a proprietary cryoprecipitation, a column fractionation on a Bio Gel A-15 M column, a ammonium sulfate dialysis, a calcium ion dissociation, a further chromatographic fractionation on a Sepharose (CL-4B column, dialysis and a final chromatographic separation on a QAE cellulose column.
Bio-rad Price List J, January 1984, pages 94-99, discloses that Bio-Gel TSK columns are suited to separating polycations or neutral polymers, such as polyacrymide (PAM), polyethylene oxide, polyvinyl alcohol (PVA) and the like. Bio-Gel TSK columns are packed with a hydroxylated polyether-based material.
T. W. Hearn et al, "HPLC Peptides and Protein", American Laboratory, Vol. 14(10), October, 1982, page 1 et seq. describes the use of high performance liquid chromatography in peptide and protein separation.